Knockin gene-mutated mouse comprising a mutant presenilin-1 gene

ABSTRACT

A gene-mutated animal such as a mouse which comprises a mutant prsenilin-1 gene comprising a DNA having a sequence encoding a mutant presenilin-1 protein in which an amino acid is substituted with a different amino acid in an amino acid sequence of a presenilin-1 protein; for example, a mutant presenilin protein in which isoleucine at position 213 is substituted with an amino acid other than isoleucine, e.g., threonine, in a mouse presenilin-1 protein. The animal is useful as an animal model which has pathological conditions closer to a human patient with Alzheimer&#39;s disease.

FIELD OF THE INVENTION

This invention relates to a trans-genic animal. More specifically, theinvention relates to a presenilin trans-genic animal with a transferredmutated presenilin gene causing human Alzheimer's disease.

BACKGROUND OF THE INVENTION

Alzheimer's disease exhibits a symptom of progressive dementia. Itspathologic histology is characterized by emergence of a huge number ofsenile plaques in the brain and accumulation of neurofibrillarydegenerations in neurons. The disease is neurodegenerative in whichneurons are gradually leading to deciduation. Alzheimer's diseasegenerally develops in old age and its prevalence is known to increasewith aging. At present, a definitive treatment of Alzheimer's disease isimpossible. Accordingly, in order to prepare for sharp increase of theold age population in the future, early developments of a method oftherapeutic and preventive treatment of Alzheimer's disease and aneffective medicament for preventive and therapeutic treatment of thedisease are desired.

Senile plaque is a deposit outside neurons which contains variousingredients, and whose main ingredient is a peptide consisting of 39–42amino acid residues called amyloid β protein (Aβ). Amyloid precursorprotein (APP) is cleaved by proteases tentatively named β secretase andγ secretase to produce amyloid β. In the senile plaque, the amyloid βdeposits as a rigid construct having β sheet structure. The senileplaque is first formed as a “stain-like” deposition called as a diffusesenile plaque. At this stage, neurodegeneration has not yet occurred. Itis considered that, as the diffuse senile plaque becomes a more rigiddeposition, the degeneration or deciduation of neurocytes occurs, whichresults in the onset of symptoms of Alzheimer's disease such asdementia. There are Aβ 40 consisting of 40 amino acid residues and Aβ 42consisting of 42 amino acid residues as main amyloid β. Most of amyloidβ generated by cells is Aβ 40, and only a little amount of Aβ 42 exists.However, Aβ 42 has higher aggregation properties, and therefore, A β42is considered to have a more significant role than Aβ 40 in theformation of senile plaque (Tamaoka, Naika (Internal Medicine), Vol. 77,P843, 1996).

In Alzheimer's disease, familial onsets are observed which exhibit anautosomal dominant inheritance. A gene first identified as a causal geneof the familial onset of Alzheimer's disease in 1991 is a mutant of APP,a gene located on chromosome 21 in which amino acid residue at position717 is mutated from valine to isoleucine (Goate A. et al., Nature, Vol.349, P704, 1991).

Other mutants of APP as causes of Alzheimer's disease were found such asthose where said amino acid residue at position 717 is mutated tophenylalanine (Murrell J. et al., Science, Vol. 254, P97, 1991); wherethe amino acid residue at the same position is mutated to glycin(Chartier, Harlin et al., Nature, Vol. 353, P844, 1991); where two aminoacid residues at positions 670 and 671 are mutated fromlysine-methionine to asparagine-leucine (Mullan M. et al., NatureGenet., Vol. 1, P345, 1992); and where amino acid residue at position692 is mutated from alanine to glycin (Hendrisk L. et al., NatureGenet., Vol. 1, P218, 1992) and the like.

Apolipoprotein E (apo E) was reported in 1993 as a causal factor or arisk factor of the familial Alzheimer's disease. Persons withAlzheimer's disease were found to have apoE4, in which the amino acidresidue at position 112 is arginine and the amino acid residue atposition 158 is arginine, at a significantly higher rate than healthypersons among isomers of apoE whose genes are located on chromosome 19(Corder E. H. et al., Science, Vole 261, P921, 1993).

After then, a mutant of the gene “presenilin-1” (PS-1, initially calledas S182) being located on chromosome 14 (Sherrington R. et al., Nature,Vol. 375, P754, 1995) and a mutant of the gene “presenilin-2” (PS-2,initially called as E5-1 or STM-2) being located on chromosome 1(Sherrington R. et al., Nature, Vol. 375, P754, 1995) were found as newcausal genes for Alzheimer's disease in 1995 (in the specification, eachgene is called as “presenilin-1 gene” and “presenilin-2 gene”,respectively, and each gene product is called as “presenilin-1 protein”and “presenilin-2 protein”, or “PS-1” and “PS-2”, respectively.)

Presenilin-1 protein and presenilin-2 protein consisting respectively of467 and 448 amino acid residues have a seven (or eight)-foldtransmembrane primary structure, and accordingly, they are presumablypresent as membrane proteins. Homology of the two proteins is high atamino acids level, i.e., 67% in total and 84% in the transmembranedomain alone. As for function of presenilin-1 protein, the protein issuggested to possibly have similar functions to nematode sel-12 proteinor SPE-4 protein because of high homology to these proteins. SPE-4protein participates in nematode spermatogenesis process and isconsidered to be involved in transport and storage of proteins.

Consequently, presenilin-1 protein is believed to participate possiblyin processing of membrane proteins such as APP, axoplasmic transport,and fusion of membrane vesicle with membranes. The sel-12 was found as agene which remedies an embryological abnormality caused by mutation oflin-12 which controls nematode development. The lin-12 is considered tobe involved in intercellular signal transduction, and accordingly,presenilin-1 protein is also suggested to possibly participate in acertain step of intercellular signal transduction.

The first report on presenilin-1 protein describes that mutationscausing the familial Alzheimer's disease are substitutions of amino acidresidues at five positions. After this report, genes mutated at varioussites were found from many families afflicted with familial Alzheimer'sdisease, which include OS-2 (isoleucine at position 213 is mutated tothreonine) and OS-3 (valine at position 96 is mutated to phenylalanine),both reported by the present inventors (Kamino K. et al., Neurosci.,Lett., Vol. 208, P195, 1996), and more than 40 types of amino acidsubstitutions have been known at more than 30 sites so far (Hardy. TINS,Vol. 20, P154, 1997).

At present, 70–80% of the familial Alzheimer's disease is believed to berelated to the mutation of presenilin-1 protein. Mutations at two siteshave been reported as for presenilin-2 protein. As explained above,genetic analysis has proved that mutants of presenilin-1 andpresenilin-2 proteins are deeply involved in the familial Alzheimer'sdisease.

Studies on mechanism how the mutants of presenilin-1 and presenilin-2proteins cause the onset of Alzheimer's disease have also beenprogressed. It has been reported that Aβ 40 is almost the same level asnormal presenilin-1 and presenilin-2 proteins, whilst Aβ 42 is highlyincreased as compared to normal presenilin-1 and presenilin-2 proteinsin serum or a culture medium of dermal fibroblasts from a patient withAlzheimer's disease having the aforementioned mutants (Scheuner D. etal.: Nature Med., Vol. 2, P864, 1996); in a culture medium of a cellline transformed by mutants of presenilin-1 protein and presenilin-2protein (Xia W. et al.: J. Biol. Chem. Vol. 272, P7977, 1997; BorcheltD. R. et al.: Neuron, Vol. 17, P1005, 1996; Citron, M. et al.: NatureMed., Vol. 3, P67, 1997); and in the brain tissue of a patient withfamilial Alzheimer's disease having the mutant presenilin-1 protein(Lemere C. A. et al.: Nature Med., Vol. 2., P1146, 1996).

These reports show that the mutants of presenilin-1 protein andpresenilin-2 protein, which cause the familial Alzheimer's disease,possibly trigger the onset of Alzheimer's disease by the increase of Aβ′42 which is considered to play a significant role in the formation ofsenile plaque. A trans-genic mouse transferred with a gene encoding themutant presenilin-1 protein was created (Duff K. et al.: Nature, Vol.383, P710, 1996, Borchelt D R. et al.: Neuron, Vol. 17, P1005, 1996 andCitron M. et al.: Nature Med., Vol. 3, P67, 1997). It was reported thatAβ 42 in the brain of the trans-genic mouse selectively increased. Theseresults are strong supports of the possibility that mutants ofpresenilin-1 protein and presenilin-2 protein causing the familialAlzheimer's disease increase Aβ 42 which possibly has significant rolesin the formation of senile plaque, thereby develop Alzheimer's disease.However, no description is given about histological study of the mouse'sbrain in the above reports on the trans-genic mouse, which presumablydue to no observation of remarkable histological change in the brain ofthe trans-genic mouse.

Generally, trans-genic animals are useful as a means of analyzingfunctions of a target gene in vivo. However, it is technically difficultto control the expression of a transferred gene quantitatively, tissuespecifically, or time specifically during development. There is also aproblem in that two different gene products are present as a mixture inthe trans-genic animals since a gene inherently possessed by the animalstill works for normal expression, and functions of a transferred genecannot be sufficiently analyzed. Furthermore, when the transferred geneis subjected to particularly excessive expression, functions notinherently performed in vivo may appear in trans-genic animals, whichresults in a defect of possible confusion in analysis of constructedgene-mutated animals.

Apart from trans-genic animals, knockout animals may also be used as ameans of analyzing functions of a target gene. In a knockout animal, atarget gene inherently possessed by the animal is artificially destroyedso as to be dysfunctional. A detailed analysis of knockout animals mayreveal functions of a target gene in vivo. However, particular changesin knockout animals created as homozygote sometimes fails to appear,since the functions of the other gene products in the knockout animalmay substitute for that of the destroyed gene products. Furthermore,there is also a problem in that an animal as homozygote may sometimes belethal because the destroyed gene product is essential to the animal'sdevelopment and growth, whilst thorough analysis of gene functions of ananimal as viable heterozygote is practically impossible.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide, for creation of ananimal pathologic model of Alzheimer's disease, an animal as apathological model whose pathologic conditions is closer to those of apatient with Alzheimer's disease, instead of a trans-genic animal havingthe aforementioned defects. More specifically, the object of the presentinvention is to provide a gene-mutated animal capable of expressing amutant presenilin protein in the brain by transfer of a mutant of apresenilin gene which is believed to be a causal gene of Alzheimer'sdisease (a mutant presenilin gene) according to a homologousrecombination technique. Further objects of the present invention are toprovide a method of producing said gene-mutated animal; a plasmid usefulfor the aforementioned production method; and a method for evaluating asubstance or an agent effective for preventive and/or therapeutictreatment of Alzheimer's disease using the aforementioned gene-mutatedanimal.

In order to reveal roles of presenilin-1 protein and mechanism of theonset of Alzheimer's disease by the mutation of presenilin-1 gene, theinventors of the present invention created a knockin mouse in whichpresenilin-1 gene inherently possessed by the mouse is replaced with theaforementioned presenilin-1 gene with OS-2 type mutation. As a result,the inventors found that the gene-mutated mouse successfully avoided thedefects with the trans-genic mice and the knockout mice, and that theanimal was useful for investigations of cause and pathology of familialAlzheimer's disease caused by the mutant presenilin-1 gene. Theinventors further continued the research, and achieved the presentinvention set out below.

The present invention thus provides a non-human gene-mutated animalhaving a mutant presenilin-1 gene, and more preferably, the inventionprovides a gene-mutated animal having a mutant presenilin-1 gene whichcomprises a DNA having a sequence encoding a presenilin-1 protein inwhich an amino acid in an amino acid sequence of a presenilin-1 proteinis substituted with a different amino acid.

The present invention also provides:

-   -   a non-human gene-mutated animal having a mutant presenilin-1        gene which comprises a DNA having a sequence encoding a mutant        presenilin-1 protein which has an amino acid sequence in which        one or more amino acids at positions selected from the group        consisting of amino acid numbers 79, 82, 96, 115, 120, 135, 139,        143, 146, 163, 209, 213, 231, 235, 246, 250, 260, 263, 264, 267,        269, 280, 285, 286, 290, 318, 384, 392, 410, 426, and 436 is        substituted with different amino acid(s) in an amino acid        sequences of a presenilin-1 protein, preferably a mouse-derived        presenilin-1 protein; and    -   a non-human gene-mutated animal having a mutant presenilin-1        gene which comprises a DNA having a sequence encoding a mutant        presenilin-1 protein which has one or more mutations selected        from the group consisting of A79V, V82L, V96F, Y115H, Y115C,        E120K, E120D, N135D, M139V, M139T, M139I, I143F, I143T, M146L,        M146V, H163Y, H163R, G209V, I213T, A231T, A231V, L235P, A246E,        L250S, A260V, C263R, P264L, P267S, R269G, R269G, R269H, E280A,        E280G, A285V, L286V, S290C, E318G, G384A, L392V, C410Y, A426P        and P436S in an amino acid sequence of a presenilin-1 protein,        more preferably a mouse presenilin-1 protein (Each alphabet        represents an amino acid expressed as a one-letter symbol, each        number represents an amino acid number from the N-terminus of        the presenilin-1 protein, and the descriptions mean that a        wild-type amino acid shown in the left of the numerical figure        is substituted with an amino acid shown in the right. In the        specification, mutant presenilin-1 protein and mutant        presenilin-2 protein are shown in the same manner.).

The present invention further provides a non-human gene-mutated animalhaving a mutant presenilin-1 gene which comprises a DNA having asequence encoding a mutant presenilin-1 protein in which isoleucine atposition 213 of a presenilin-1 protein is substituted with an amino acidother than isoleucine, and a non-human gene-mutated animal having amutant presenilin-1 gene which comprises a DNA having a sequenceencoding a mutant presenilin-1 protein in which isoleucine at position213 of a presenilin-1 protein is substituted with threonine.

According to preferred embodiments of the aforementioned inventions,there are provided:

-   -   the aforementioned gene-mutated animal having a mutant        presenilin-1 gene wherein a DNA sequence encoding around an        amino acid at position 213 in an amino acid sequence of a        presenilin-1 protein is mutated to the following sequence:

-   5′-TGTGGTCGGGATGATYGCC AVC CACTGGAAAGGCCC-3′ (SEQ ID NO: 18)    wherein V represents a base other than T, Y represents T or C, and    the underlined bases encode the amino acid at position 213;    -   the aforementioned gene-mutated animal having a mutant        presenilin-1 gene wherein a DNA sequence encoding around an        amino acid at position 213 in an amino acid sequence of a        presenilin-1 protein is mutated to the following sequence:

-   5′-TGTGGTCGGGATGATYGCC ACC CACTGGAAAGGCCC-3′ (SEQ ID NO: 19)    wherein Y represents T or C, and the underlined bases encode the    amino acid at position 213; and    -   the aforementioned gene-mutated animal having a mutant        presenilin-1 gene wherein a DNA sequence encoding around an        amino acid at position 213 in an amino acid sequence of a        presenilin-1 protein is mutated to the following sequence:

-   5′-TGTGGTCGGGATGATYGCC NNN CACTGGAAAGGCCC-3′ (SEQ ID NO: 20)    wherein each N independently represents A G, T or C and NNN    represents a codon as triplet bases which encodes an amino acids    other than isoleucine, Y represents T or C, and the underlined bases    encode the amino acid at position 213.

From another aspect, the present invention provides a non-humangene-mutated animal having a mutant presenilin-2 gene which comprises aDNA having a sequence encoding a protein in which an amino acid atposition 141 and/or 436 is substituted with a different amino acid in anamino acid sequence of a presenilin-2 protein. As a preferred embodimentof the invention, there is provided the aforementioned non-humangene-mutated animal wherein the mutant presenilin-2 gene comprises a DNAhaving a sequence encoding a mutant presenilin-2 protein which containsa mutation of N141I and/or M239V in an amino acid sequence of apresenilin-2 protein.

As preferred embodiments of the aforementioned gene-mutated animals, thepresent invention provides the aforementioned gene-mutated animalwherein overexpression of amyloid β protein is caused by the mutantpresenilin-1 gene and/or the mutant presenilin-2 gene; theaforementioned gene-mutated animal which can express the mutantpresenilin protein and wherein the expression of said protein inducesthe production of amyloid β protein in an amount sufficient to form aprogressive neural disease in a peripheral portion of the cerebralcortex of the brain of the animal; the aforementioned gene-mutatedanimal wherein the animal is a rodent, preferably a mouse; theaforementioned gene-mutated animal wherein the aforementioned mutantpresenilin-1 gene and/or the aforementioned mutant presenilin-2 gene aretransferred by homologous recombination; the aforementioned gene-mutatedanimal wherein amount of the amyloid protein expression in a braintissue induced by the aforementioned presenilin-1 gene is sufficient tocause affected behavior in a memory learning test in comparison with anormal animal, and to induce abnormal neuropathy in a peripheral portionof the cerebral cortex of the hippocampus of the brain of the animal;and the non-human gene-mutated animal having a DNA which comprises amutant preceilin-1 gene encoding a mutant preceilin-1 protein in whichone or two or more amino acids is substituted with a different aminoacid in the amino acid sequence of the presenilin-1 protein togetherwith a DNA having a nucleotide sequence encoding a marker protein.

From further aspect, the present invention provides a plasmid comprisinga DNA having a sequence of a mutant presenilin-1 gene wherein a DNAsequence encoding around an amino acid at position 213 of a presenilin-1protein is the following sequence:

-   5′-TGTGGTCGGGATGATYGCC AVC CACTGGAAAGGCCC-3′ (SEQ ID NO: 18)    wherein V represents A, G, or C, Y represents T or C, and the    underlined bases encode an amino acid at position 213; and-   a plasmid comprising a DNA having a sequence of a mutant    presenilin-1 gene which encodes a mutant presenilin-1 protein    wherein an amino acid at position 213 is substituted with an amino    acid other than isoleucine in an amino acid sequence of the    presenilin-1 protein and a DNA sequence encoding around the amino    acid at position 213 of presenilin-1 protein is the following    sequence:-   5′-TGTGGTCGGGATGATYGCC NNN CACTGGAAAGGCCC-3′ (SEQ ID NO: 20)    wherein Y represents T or C, each N independently represents A, G,    T, or C and NNN represents a codon as triplet bases encoding an    amino acid other than isoleucine, and the underlined bases encode    the amino acid at position 213. Additionally, the present invention    also provides a chromosomal DNA containing exon 8 of a mutant    presenilin-1 gene encoding a mutant presenilin-1 protein wherein an    amino acid at position 213 is substituted with an amino acid other    than isoleucine in an amino acid sequence of a presenilin-1 protein.

Furthermore, the present invention provides a plasmid comprising a DNAwherein a Sau3AI site is introduced into a nucleotide sequencecomprising the whole or a mutated part of a cDNA or chromosomal DNA of amutant presenilin-1 gene encoding a mutant presenilin-1 protein in whichan amino acid at position 213 is substituted with an amino acid otherthan isoleucine in an amino acid sequence of presenilin-1 protein. Alsoprovided are the aforementioned plasmid wherein the substitution of theamino acid is isoleucine at position 213 with threonine; and a plasmidcomprising a DNA specified by the following nucleotide sequence:

-   5′-TGTGGTCGGGATGATYGCCACCCACTGGAAAGGCCC-3′ (SEQ ID NO: 19)    wherein Y represents T or C.

In addition to the above inventions, the present invention also providesa gene encoding a mouse mutant presenilin-1 protein wherein isoleucineat position 213 is substituted with an amino acid other than isoleucinein an amino acid sequence of a mouse presenilin-1 protein; and theaforementioned gene wherein the substitution is from isoleucine tothreonine. Also provided are a plasmid comprising: (1) a gene encoding amouse mutant presenilin-1 protein wherein isoleucine at position 213 issubstituted with an amino acid other than isoleucine in an amino acidsequence of a mouse presenilin-1 protein; and (2) a neomycine expressionunit flanked by loxPs; and the aforementioned plasmid wherein thesubstitution is from isoleucine to threonine (loxP has been disclosed inJapanese Patent Laid-Open Publication (Kohyo) No. 4-501501, page 4).

From further aspect, the present invention provides an embryo introducedwith a plasmid comprising a DNA represented by the nucleotide sequence:

-   5′-TGTGGTCGGGATGATYGCCACCCACTGGAAAGGCCC-3′ (SEQ ID NO: 19) wherein Y    represents T or C; an embryo obtained by homologous recombination    using each of the aforementioned plasmids; and the aforementioned    embryo derived from a mammalian rodent, more preferably from a    mouse. The invention also provides a primary cell culture or    subcultured cell obtained by isolating a cell from the    aforementioned gene-mutated animal and culturing the cell by tissue    culture; a method for producing a non-human gene-mutated animal    wherein the method comprises the step of transferring a mutant    presenilin-1 gene by homologous recombination into an embryo of an    animal, wherein the mutant presenilin-1 gene is capable of    expressing the mutant presenilin-1 and inducing production of    amyloid β protein in an amount sufficient to form a progressive    neural disease in a peripheral portion of the cerebral cortex of the    brain; and the aforementioned production method wherein a mutant    presenilin-1 protein can be expressed wherein isoleucine at position    213 is substituted with an amino acid other than isoleucine.

Additionally, the invention provides a method for evaluating a substanceuseful for therapeutic and/or preventive treatment of Alzheimer'sdisease which comprises the step of subjecting the aforementionedgene-mutated animal which is administered with a test substance to acomparison with the gene-mutated animal not administered with the testcompound. A typical example of the method for evaluation includes ascreening method. According to preferred embodiments of the invention,there are provided the aforementioned method for evaluation wherein thecomparison is conducted by using a memory learning test; theaforementioned method for evaluation wherein the comparison is conductedby using a pathological test; the aforementioned method for evaluationwherein the comparison is conducted by a pathological test based onneuropathology in a peripheral portion of the cerebral cortex; theaforementioned method for evaluation wherein the comparison conducted bythe pathological test based on neuropathology is a comparison of one ormore items selected from the group consisting of suppression of decreasein overgrown gliosis in a peripheral portion of the cerebral cortex ofthe brain, suppression of decrease in uptake of 2-deoxyglucose in aperipheral portion of the cerebral cortex of the brain, and suppressionof decrease in availability of 2-deoxyglucose in the cerebral cortex ofthe brain; and the aforementioned method for evaluation wherein thecomparison is conducted for one or more items selected from the groupconsisting of survival period of time, exploratory behavior andmigratory behavior.

Still further, the present invention provides a method for evaluating amedicament for therapeutic and/or preventive treatment of Alzheimer'sdisease which comprises the step of culturing a primary cell culture ora subcultured cell in vitro in the presence of a test compound; a methodfor diagnosing Alzheimer's disease or a possibility of onset ofAlzheimer's disease, which comprises the use of a partial nucleotidesequence of a mutant presenilin-1 gene encoding an OS-2 type mutantpresenilin-1 protein; a substance useful for therapeutic and/orpreventive treatment of Alzheimer's disease selected by each of theaforementioned evaluation methods; and a medicament for therapeuticand/or preventive treatment of Alzheimer's disease comprising theaforementioned substance as an active ingredient.

The present invention also provides a gene-mutated animal having amutant presenilin gene and a gene encoding a mutant amyloid precursorprotein, wherein the animal is a hybrid animal or its progeny which isproduced by mating the aforementioned gene-mutated animal with an animalhaving a gene encoding a mutant protein of the amyloid precursor proteinand a high productivity of amyloid β protein, and more preferably theanimal is a hybrid mouse or its progeny which is produced by the matingor which is born as a result of the mating. According to a preferredembodiment of the invention, there is provided the aforementionedgene-mutated animal wherein the animal having a gene encoding a mutantprotein of the amyloid precursor protein and a high productivity ofamyloid β protein is a PS1-mutated mouse.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a restriction map of a chromosomal DNA fragment Pα containingexon 8 of mouse presenilin-1 which was obtained by cloning from a mousegenomic DNA library.

FIG. 2 illustrates a scheme of the construction method of plasmid pmX-1containing a partial region of exon 8 of the mouse presenilin-1 genewhich comprises a region introduced with an OS-2 type mutation by asite-directed mutation technique.

FIG. 3 illustrates a process of preparing a targeting vector.

FIG. 4 illustrates a process of preparing a targeting vector.

FIG. 5 illustrates a process of preparing a targeting vector.

FIG. 6 illustrates a process of preparing a targeting vector.

FIG. 7 illustrates a process of preparing a targeting vector and thestructure of the targeting vector pOS-2 neoloxP.

FIG. 8 illustrates results of electrophoresis on 1% agarose gel of thePCR product obtained by mating #2 mouse (male) having OS-2 mutantpresenilin-1 gene with F4 of CAG-cre#13 mouse (female), cutting a smallpiece off from the resulting progeny's tail, obtaining chromosomal DNAfrom the specimen, and carrying out PCR according to the methoddescribed in Example 10. It is shown that mice correspond to 2nd and 4thlanes from the right have no neo expression unit on their chromosomalDNA. In the figure, the leftmost lane shows a molecular weight marker.[A] indicates bands showing neo deficiency on the chromosomal DNA, [B]indicates bands showing that the chromosomal DNA is the wild type, and[C] indicates is bands showing the existence of neo on the chromosomalDNA.

BEST MODE FOR CARRYING OUT THE INVENTION

A mutant presenilin gene used in the production of the gene-mutatedanimal of the present invention is a gene encoding a mutant presenilinprotein, and as used herein, “mutant presenilin gene” means either of,or both of a mutant presenilin-1 gene or a mutant presenilin-2 gene and“mutant presenilin protein” means either of, or both of a mutantpresenilin-1 protein or a mutant presenilin-2 protein. The mutantpresenilin gene has the property of increasing the production of amyloidβ protein. The gene-mutated animal of the present invention is a mammaltransferred with the above-mentioned mutant presenilin gene for exampleby homologous recombination. The mutation existing in the mutant proteinis preferably a result of substitution of an amino acid residue. Thenumber of mutations is not limited, and may preferably be 1.

The full length sequence of a mammal-derived preselin-1 protein isdescribed in, for example, E. Levy-Lahad, et al., Science, 269, pp.973–977, 1995. The full-length sequences of human and mouse presenilin-1proteins and examples of DNA sequences that encode the proteins areshown in the sequence listings as SEQ ID NOS: 1 to 4. For example, inthe mouse-derived presenilin-1, mutation sites may preferably be one ormore sites selected from No. 79, No. 82, No. 96, No. 115, No. 120, No.135, No. 139, No. 143, No. 146, No. 163, No. 209, No. 213, No. 231, No.235, No. 246, No. 250, No. 260, No. 263, No. 264, No. 267, No. 269, No.280, No. 285, No. 286, No. 290, No. 318, No. 384, No. 392, No. 410, No.426, and No. 436.

More preferable mutations are one or more mutations selected from thegroup consisting of A79V, V82L, V96F, Y115H, Y115C, E120K, E120D, N135D,M139V, M139T, M139I, I143F, I143T, M146L, M146V, H163Y, H163R, G209V,I213T, A231T, A231V, L235P, A246E, L250S, A260V, C263R, P264L, P267S,R269G, R269G, R269H, E280A, E280G, A285V, L286V, S290C, E318G, G384A,L392V, C410Y, A426P, and P436S in the amino acid sequence of thepresenilin-1 protein, more preferably in the amino acid sequence of themouse-derived presenilin-1 protein. Among these mutations, the mutationwherein the amino acid at position 213 is substituted with another aminoacid (referred to in some cases as “OS-2 type mutation” in thespecification) is a particularly preferable mutation. For example, amutation wherein isoleucine at position 213 is substituted with an aminoacid other than isoleucine, or a mutation wherein isoleucine at position213 is substituted with threonine is most preferable.

The full-length sequence of a mammal-derived preseline-2 protein isdescribed in, for example, Science, 269, pp. 973–977, 1995. Position 141and/or position 436 are preferable mutation sites, and in themouse-derived sequence N141I and/or M239V are more preferable. One ormore mutations may exist in either of presenilin-1 protein orpresenilin-2 protein, or both of the proteins.

The gene-mutated animal of the present invention is characterized byhaving the above mutant presenilin-1 gene and/or mutant presenilin-2gene on its chromosomal DNA. The gene-mutated animal is not limited sofar that the animal is a mammal and a kind of the animal is notparticularly limited. For example, a rodent may suitably be used. Amouse is particularly preferred. The gene-mutated animal of the presentinvention can be produced by constructing a plasmid using a DNA having asequence of about 10 kbp comprising a mutant presenilin gene, and thentransferring the plasmid into an embryonic stem cell and thereby causinghomologous recombination intracellularly.

The gene-mutated animal of the present invention is characterized inthat the amino acid mutation occurs mostly at only one position due tothe transfer of the aforementioned mutant presenilin-1 and/orpresenilin-2 gene by homologous recombination. In the case of aso-called “trans-genic animal”, a DNA sequence comprising a mutantportion is inserted randomly into chromosomal DNA, and tens of copies ofa repeated sequence are inserted at plural sites. The gene-mutatedanimal of the present invention can avoid the problems, and it ispossible to accurately analyze pathology of Alzheimer's disease atgenetic level. Where a DNA comprises a marker or the like is transferredto the gene-mutated animal of the present invention, the animal may havea site of the marker and a sequence for insertion of the marker. Forexample, for insertion at a site capable of being cleaved with Sau3AI,one nucleotide can be substituted, and such substitution can be verifiedby cleaving a PCR product with Sau3AI, followed by subjecting thefragments to electrophoresis or the like.

The gene-mutated animal of the present invention has a characteristicfeature of producing amyloid β protein in a larger amount in comparisonwith a normal animal due to the genetic mutation. An increased amount ofamyloid β protein achieved by the gene-mutated animal of the presentinvention is not particularly limited, and the amount may preferably besufficient for recognition of a substantial difference in the evaluationof degrees of memory disorder, pathological observations, and variousneural disorders as compared to a normal animal.

DNAs, plasmids, cell cultures, and embryos of mammalian cells providedby the present invention are characterized to have a mutant presenilin-1gene and/or a mutant presenilin-2 gene. For example, a cDNA or afull-length chromosomal DNA of a mutant presenilin-1 gene encoding themutant presenilin-1 protein, preferably an OS-2 type mutant presenilin-1protein, or the DNA sequence comprising one or more mutation sites; aplasmid comprising a DNA being the above cDNA or full length chromosomalDNA, or the above DNA comprising one or more mutation sites, which isfurther introduced with an Sau3AI site; a chromosomal DNA comprisingexon 8 of a mutant presenilin-1 gene encoding an OS-2 type mutantpresenilin-1 protein fall within the present invention. Further, thepresent invention encompasses the above gene or the DNA which furthercomprises one or more, preferably 1 to 20, more preferably 1 to severalsubstitutions of bases.

Examples of DNAs and plasmids of the present invention include, forexample:

-   -   1) a DNA comprising a mutant presenilin-1 gene encoding a mutant        presenilin-1 protein wherein isoleucine at position 213 of the        presenilin-1 protein is substituted with threonine, or a plasmid        comprising said DNA;    -   2) a DNA comprising a mutant presenilin-1 gene wherein a DNA        nucleotide sequence encoding amino acids around position 213 of        the amino acid sequence of a mutant presenilin-1 protein is the        following sequence:

-   5′-TGTGGTCGGGATGAT Y GCCA V CCACTGGAAAGGCCC-3′ (SEQ ID NO: 18)    wherein V represents a nucleotide other than T and Y represents T or    C, or a plasmid comprising said DNA;    -   3) a DNA comprising a mutant presenilin-1 gene wherein a DNA        nucleotide sequence encoding amino acids around position 213 of        the amino acid sequence of an OS-2 type mutant presenilin-1        protein is the following sequence:

-   5′-TGTGGTCGGGATGAT Y GCC NNN CACTGGAAAGGCCC-3′ (SEQ ID NO: 20)    wherein Y represents T or C, each N independently represents A, G,    T, or C and NNN represents a codon as triplet bases encoding an    amino acid other than isoleucine, or a plasmid comprising said DNA;    -   4) Any one of the DNAs or plasmids comprising said DNAs        according to the aforementioned 1) to 4) wherein a Sau3AI        restriction site is introduced;    -   5) a DNA or a plasmid comprising said DNA wherein a Sau3AI        restriction site is introduced into a sequence comprising the        full-length of a cDNA or a chromosomal DNA of a mutant        presenilin-1 gene encoding a mutant presenilin-1 protein wherein        isoleucine at position 213 is substituted with threonine in an        amino acid sequence of presenilin-1 protein, or into a mutated        portion of said sequence;    -   6) a DNA comprising exon 8 of a mutant mouse presenilin-1 gene        encoding an OS-2 type mutant presenilin-1 protein and a neomycin        expression unit flanked by loxP, or a plasmid comprising said        DNA; and,    -   7) a DNA comprising exon 8 of a mutant presenilin-1 gene        encoding a mutant presenilin-1 protein wherein isoleucine at        position 213 is substituted with threonine in an amino acid        sequence of presenilin-1 protein and a neomycin expression unit        flanked by loxP, or a plasmid comprising said DNA. However, the        scope of the invention is not limited to these specific        examples:

The embryos or the cells provided by the present invention includes anembryo or a cell into which the above plasmid, e.g. a plasmid comprisinga PRL-104 or PRL-105 nucleotide sequence is transferred. Preferablecells of the present invention include those transferred with a geneencoding a mutant presenilin protein which comprises a mutation atposition 213 of the amino acid sequence of presenilin-1 protein byhomologous recombination using the aforementioned plasmid. Sorts of theembryos or cells are not limited so far that they are derived from amammal, and those derived from a rodent, preferably a mouse may be used.

Production of the Gene-Mutated Animal

After the DNA encoding a mutant of human presenilin is obtained, thepresenilin gene mutated animal of the present invention can be producedaccording to the process described below. An example will be explainedwherein a mouse is used as an mammal and the human mutant humanpresenilin-1 gene is used as the mutant human presenilin gene. However,the gene-mutated animal of the present invention is not limited to thoseproduced by using these materials. Further, this method is one exampleof the method of production of the gene-mutated animal of the presentinvention and the method of the present invention is not limited to thefollowing method. By referring to the general method described below andspecific methods described in the examples, and by suitably modifying oraltering these methods as required, a person skilled in the art canreadily produce the gene-mutated animal of the present invention.

In order to prepare a probe for use in the PCR method, a DNA fragment,which comprises a site for mutation in exon 8 of a presenilin-1 genederiving from an animal to be used for the production, is obtained froma mouse genomic DNA library. A mouse genomic DNA library of any strainmay be used, including a mouse genomic library from mouse 129 straindescribed in the examples. Where a mouse is used as an animal forintroduction of mutation, exon 8 of mouse presenilin-1 gene is used.Where other sort of animal is used, it is necessary to select anappropriate segment.

After of the DNA fragment prepared by the above process is labeled (³²P)by random priming, screening of the genomic library is performed usingthe labeled probe, and a chromosomal DNA fragment comprising exon 8 ofthe presenilin-1 gene is then cloned. A portion for mutation in exon 8of the cloned presenilin-1 gene is further subcloned, and then amutation is introduced.

A targeting vector is constructed which comprises the chromosomal DNAcomprising exon 8 of the mouse presenilin-1 gene into which the mutationwas introduced. As a selective marker, neo expression unit is introducedinto the targeting vector to facilitate that cells whose chromosome isnot introduced with the vector are killed by the addition of G418 (anantibiotic) to a medium. After the targeting vector is introduced intoan ES cell by means of electroporation or by another method for genetransfer into a cell, the ES cells are cultured in the presence of G418and colonies formed are collected. Each of the colonies obtained isdivided into two portions. One portion is preserved by culturing,sub-culturing, or freezing. The other portion is used to investigate EScells into which a desired mutation in exon 8 of the mouse presenilin-1gene is introduced by homologous recombination, are examined. Thepreserved portion of the colony of the ES cells with the desiredmutation introduced is taken and used in the process below.

From a pregnant mouse, an embryo at the 8-cell stage is removed. Theembryo is sprinkled with about 20 of the above-mentioned preserved EScells, and then introduced into the uterus of a pseudopregnant femalemouse. From among the born young, mice of chimeric coat color areselected. The chimeric mouse is mated with a mouse C57BL/6 strain, and amouse having the desired mutation can be obtained by the selection ofthose with agouti coat color from among the born young. The resultingmouse is heterozygote in relation to the presenilin-1 gene introducedwith the mutation, whereas the presenilin-1 gene on the other chromosomeis a wild type with no mutation.

As starting materials for preparing the probe for the cloning of thechromosomal DNA comprising exon 8 of the mouse presenilin-1 gene fromthe mouse genomic DNA library, a cDNA of a presenilin-1 gene, which isderived from a mammal other than mouse or human and whose nucleotidesequence has been known, may be used as well as those specificallymentioned in the Examples. As methods for obtaining the DNA fragmentused as the probe, a method for a large scale preparation of a plasmid,which comprises a mouse chromosomal DNA comprising a regioncorresponding to exon 8 of the mouse presenilin-1 gene in chromosomalDNA, or a cDNA of a presenilin-1 gene derived from a mammal other thanmouse or human or the like whose nucleotide sequence has been know, canbe applied as well as amplification by PCR described in the Examples.Furthermore, after the plasmid is cleaved by restriction enzymes, adesired DNA fragment can be obtained by separating a portion used as theDNA fragment by means of agarose gel electrophoresis and the like.

As a method for labeling the DNA fragment, methods such as thoseutilizing PCR in the presence of ³²P-dNTP may be used as well as therandom priming method described in the Examples. Further, labeling maybe introduced by PCR or random priming using a pre-labeledoligodeoxynucleotide as a primer. For the labeling, chemiluminescenceusing Biotin-Avidin or alkalinephosphatase or the like may also be used,as well as radioisotopes explained in the examples. An RNA fragmentlabeled by using T3 or T7 RNA polymerase may also be used as a probe.Various methods for preparing a probe are known other than thosementioned above, and a desired probe may be obtained by any method.

For introducing a desired mutation in a DNA, methods specificallydescribed in the Examples can be applied. In addition, a plasmid derivedfrom a bacteriophage such as M13 or a plasmid duplicated using ungEscherichia coli is bound complimentarily with an oligodeoxynucleotidesynthesized for introducing a mutation at a desired mutation site (basesof the site to be introduced with the mutation are not complimentary),and the resulting complex is used as a primer to prepare a heteroduplexDNA plasmid using a DNA polymerase, and then Escherichia coli (ung⁺) istransformed with the resulting plasmid to obtain a plasmid having adesired mutation. Another method (cassette method) is applied for toobtain a plasmid having a desired mutation, which comprises the steps ofsynthesizing two oligodeoxynucleotide, which have modified bases tointroduce a desired mutation, and are capable of annealing in a mutuallycomplimentary manner and designed to give restriction enzyme sites atboth terminals, and ligating the oligodeoxynucleotide to a plasmid forintroduction of a mutation using DNA ligase. By appropriately modifyingor altering the above methods depending on a purpose, the object maysometimes be more effectively achieved. In addition, as method forintroducing a mutation, various methods available in the art are known,and accordingly, any method can be applied to achieve the object.

The targeting vector may preferably comprise a selective markerexpression unit as an essential element which comprises a mousechromosomal DNA fragment introduced with a mutation, a DNA fragmentencoding a selective marker, a promoter for controlling transcriptionthereof, and a terminator. The mouse chromosomal DNA fragment introducedwith a mutation is a necessary portion for causing homologousrecombination in the ES cell, and the mouse chromosomal DNA fragmentsflanking the position of the mutation at both sides are also necessary.The target vector thus has a DNA fragment in which only the mutatedbases are different from a native mouse chromosomal DNA. The length ofthe fragment may preferably about 10 kbp, and generally some degree oflengthening or shortening is permissible. However, where the fragment istoo short, frequency of homologous recombination may sometimes belowered.

As selective markers, positive selective markers such asneomycin-resistant gene and hygromycin-resistant gene, and negativeselective markers such as thymidine kinase gene of herpes simplex virusand fragment A of diphtheria toxin are known. Any of markers used forcell culture may be used in ES cells. Where a negative selective markeris used, it is necessary to insert the marker outside the mousechromosomal DNA fragment of the targeting vector. Where a positiveselective marker is used, it is necessary to insert the expression unitin an intron in the mouse chromosomal DNA fragment of the targetingvector. When a positive marker is inserted in an exon, the inserted genegenerally loses function, and a mouse cannot be sometimes produced whichis to be produced for examination of effects of the mutation as anultimate purpose.

As an ES cell line, cell lines deriving from mouse 129 strain arefrequently used. As ES cells deriving from the above mouse strain, EScells such as D3, CCE, J1, and AB1 may be used as well as R1 describedin the Examples. For example, mouse-derived ES cells such as fromC57BL/6 mouse strain may also be used other than those from 129 strain.As methods for the introduction of the targeting vector into ES cells,electroporation as described in the Examples may generally applied. Anymethod may be used so far that the method is usable for the introductionof a plasmid into a cultured cell line, such as Ca phosphatecoprecipitation or a liposome method. When ES cells introduced with thetargeting vector are cultured in the presence of a selective marker, EScells that survive and form colonies are possibly received homologousrecombination. As a method for determining whether homologousrecombination occurs in the ES cells that form the colonies, PCR istypically used. A DNA fragment, an RNA fragment, syntheticoligodeoxynucleotide, antibody or the like that is usable as a probe maybe employed.

After ES cells are mixed with a fertilized egg at an early stage ofdevelopment and then development is continued, a mouse from a sperm oran ova deriving from the ES cell can be obtained. To mix the ES cells inwhich homologous recombination occurs with the fertilized egg at anearly stage of development, a method explained in the Examples may beapplied. In addition, a method may also be applied which comprises thesteps of removing a fertilized egg at blastula stage from a pregnantmouse, injecting 10 to 20 ES cells to the egg using an injectionpipette, transplanting the treated egg into the uterus of apseudopregnant mouse, and then continuing development to obtain theyoung.

The fertilized egg at an early stage of development for the use ofmixing with the ES cells may be eggs obtained from any strain of mouse.In order to facilitate determination whether or not ES cells isincorporated into the progeny, it is preferable to use a fertilized eggfrom a mouse strain that has a coat color different from that of a mousestrain from which the ES cells are derived. For example, the ES cellsused in the Examples are of agouti-colored 129-strain and the mouse fromwhich the fertilized egg is derived (C57BL/6) has a black colored coat.Using these materials, it is possible to easily select the young whichhave cells derived from the ES cells by selecting the young with chimeracoat color from among the born young. In this case, the young with highproportion of agouti color are most likely to have germ cells derivedfrom the ES cells. The pseudopregnant mouse may be of any strain ofmouse.

The mouse used to obtain a mouse with the desired introduced mutationthrough mating with the resulting chimera mouse may preferably a mouseof a strain with a coat color different from that of a mouse of a strainfrom which the ES cells are derived. Normally a male chimera mouse ismated with a female of a different strain, and if agouti colored youngare obtained, the resulting mice have the desired mutation asheterozygous state. Since a mouse possessing an OS-2 type mutantpresenilin-1 gene has a neo expression unit flanked by loxP sequences,it is possible to obtain a mouse in which the neo expression unit isremoved can be obtained through mating with a trans-genic mouse with atransferred cre gene.

As explained in the prior art of the present specification, it isbelieved that a mutation of presenilin-1 protein and presenilin-2protein promotes the formation of senile plaque due to an increase in Aβ42 and thereby triggers the onset of Alzheimer's disease. Amongtrans-genic mice introduced with a gene encoding the mutant APP causingfamilial Alzheimer's disease, some mice are reported to produce amyloiddeposition in the brain (Games D., et al., Nature, Vol. 373, p. 523,1995, Hsiao K. et al., Science, Vol. 274, p. 99, 1996, Sturchler-PierratC. et al., Proc. Natl. Acad. Sci. U.S.A. Vol. 94, No. 24, p. 13287,1997). In these trans-genic mice, it is considered that amyloiddeposition is induced by the increase of the amount of Aβ production inthe brain.

By mating a trans-genic animal which is transferred with a gene encodinga mutant APP and capable of forming amyloid deposition in the brain (theanimal may be homozygous or heterozygous with reference to thetransferred gene) with a PS1 gene-mutated animal of the presentinvention (the animal may be homozygous or heterozygous with referenceto the transferred gene), a hybrid animal can be produced. The animal ispreferably as mouse. For mating, either of the above animals may bemale.

A portion of the tail of the progeny is collected and chromosomal DNA isextracted. PCR is conducted by using the extracted chromosomal DNA as asubstrate and by using as primers two oligodeoxynucleotides each havinga nucleotide sequence designed to flank the mutation site of a geneencoding the mutant APP and two oligodeoxynucleotides having anucleotide sequence designed to flank the mutation site of the mutantPS1 gene.

It is possible to determine whether or not the gene encoding the APPmutant and the mutant PS1 gene of the present invention are incorporatedin an extracted chromosomal DNA by carrying out agarose gelelectrophoresis of a PCR product, and then observing, for example,presence or absence of bands and mobility of the bands in the gel, andexamining the band with the mutation by means of hybridization using anoligodeoxynucleotide having a nucleotide sequence comprising themutation. PCR may be conducted according to the method described inExample 8. Nucleotide sequences of the oligonucleotides used as the PCRprimers may be any sequences so long as they are capable of detectingthe gene encoding the APP mutant or the mutant PS1 gene. Based on theresults of PCR, an animal having both of the genes each in heterozygousstate can be obtained by selection of animals having the gene encodingthe APP mutant and the mutant PS1 gene of the present invention.

In order to obtain animals having both of the gene encoding APP mutantand the mutant presenilin-1 gene of the present invention each inhomozygous state, an individual animal having both of the genes inhomozygous state is selected from the young obtained by mating asuitable male and female selected from the animals having both genes inheterozygous state. To confirm possession of the gene encoding the APPmutant in homozygous state, a potion of the tail of the progeny is takenand chromosomal DNA is extracted, and after the cleavage of theextracted chromosomal DNA with restriction enzyme, electrophoresis isconducted using agarose gel or acrylamide gel. The DNA is then blottedonto a membrane filter, and Southern blotting is performed using as aprobe an oligodeoxynucleotide having a sequence which enables bindingspecifically to a gene encoding the APP mutant, and then density of theresulting bands are measured.

Similarly to the above process, possession of the mutant presenilin-1gene of this invention in a homozygous state can be verified.Oligodeoxynucleotides used as probes in Southern blotting can be usedafter being labeled with means ordinarily used in Southern blotting suchas a radioactive isotope and a fluorescent dye. A mouse having both ofthe gene encoding the APP mutation and the mutant presenilin-1 gene ofthe present invention can thus be produced. A hybrid mouse produced bythe above method is characterized by higher productivity of amyloid βprotein in the brain and promoted amyloid deposition.

Using the gene-mutated animal, the cells transferred with the mutantpresenilin gene, the plasmid comprising the mutant presenilin gene andthe like, it is possible to screen substances useful for preventiveand/or therapeutic treatment of Alzheimer's disease and to evaluatetheir utility. Accumulation of amyloid β in a healthy mammal progressesvery slowly, whereas the gene-mutated animal of the present inventionhas a characteristic feature of higher productivity of amyloid β.Therefore, by administering variety of test substances to thegene-mutated animal of the present invention, and comparing the animalwith non-administered animals or animals administered with a controlsubstance, it is possible to evaluate substances useful for preventiveand/or therapeutic treatment of Alzheimer's disease. A typical exampleof the evaluation includes a screening of test substances, andconditions, pathological observations, pharmacological tests and thelike can be applied as examinations.

Where the cells of the present invention are used, cells are isolatedfrom the animal of the present invention for the use as a primary cellculture, and then the cells can be stabilized and made into asubcultured cell line by immortalizing the cells of primary culture bytreatment with a virus or the like, subculturing the cells by isolatinga portion of the culture and subjecting to further cultivation in afresh tissue culture medium. The cells of the present inventionencompass the primary cell culture such as nerve cells isolated from thegene-mutated animal, as well as subcultured cells, i.e., so-called celllines, obtained by subculturing the primary culture. When a nerve cellis used as the cell of the present invention, the cell expresses a largeamount of amyloid β protein due to a result of the expression of mutantpresenilin-1 protein by the cell. Substance which prevent or delay thenerve cell death related to accumulation of amyloid β can be screenedand utility thereof can also be evaluated by adding a test substance toan in vitro culture system of such nerve cells, and comparing, forexample, cell survival period or surviving cell number after a certainperiod of time.

EXAMPLES

The present invention will be more specifically explained by way ofexamples. However, scope of the present invention is not limited tothese examples. In the following examples, presenilin-1 gene isoccasionally referred to as PS-1.

Example 1 Cloning of Chromosomal DNA containing Exon 8 of MousePresenilin-1 (PS-1) Gene

To construct a probe for isolating a chromosomal DNA containing exon 8of the mouse PS-1 gene, the following two oligodeoxynucleotides weresynthesized:

-   -   PR-8-U: 5′-GGAATTTTGGTGTGGTCGGGATGAT-3′ (SEQ ID NO: 5) (25-mer)    -   PR-8-L: 5′-GGTCCATTCGGGGAGGTACTTGA-3′ (SEQ ID NO: 6) (23-mer)

PCR was carried out by using these two oligodeoxynucleotides as PCRprimers and DNA extracted from 129 SVJ mouse genomic library(Stratagene) to obtain amplified DNA fragment of approximately 130 bp.The fragment was then labeled by random priming method in the presenceof ³²P-dCTP and then used as probes for screening of the 129 SVJ mousegenomic library. The resulting positive phage clones were examined andconfirmed that they carried the desired chromosomal DNA including exon 8of the mouse PS-1 gene. The cloned chromosomal DNA was designated as Pαand subjected to restriction mapping (FIG. 1).

Example 2 Construction of Plasmid for Introducing Mutation

DNA was extracted from the cloned phage carrying Pα and cleaved with SalI, and then subjected to electrophoresis on 1.0% agarose gel to collectPα. After the cleavage with Pst I and Xba I, the product was subjectedto electrophoresis on 1% agarose gel to collect a DNA fragment ofapproximately 600 bp including a nucleotide sequence encoding isoleucineat position 213 of mouse PS-1. The resulting DNA fragment was designatedas X-1. X-1 was ligated using T4 ligase to the plasmid pBluescript IIKS+ (Stratagene) which was cleaved beforehand with PstI and Xba I, andthen used to transform Escherichia coli to obtain plasmid pX-1.

Example 3 Introduction of OS-2 Type Mutation

An OS-2 type mutation and a Sau3A I restriction site were newlyintroduced into the plasmid pX-1 using the following twooligodeoxynucleotides PRL-104 and PRL-105. Both PRL-104 and PRL-105 were36-mers and complementary to each other:

-   PRL-104: 5′-TGTGGTCGGGA TGATC* GCCA C CCACTGGAAAGGCCC-3′ (SEQ ID NO:    7)-   PRL-105: 5′-GGGCCTTTCCAGTGG G TGGCG* ATCATCCCGACCACA-3′ (SEQ ID NO:    8)

(The underlined base is changed from a wild-type base to introduce theOS-2 type mutation, i.e., T for PRL-104 and A for PRL-105 in wild types.Asterisked bases are changed from wild-type bases to introduce theSau3AI site, i.e., T for PRL-104 and A for PRL-105 in wild types.)

The introduction of the mutation was carried out by using QUICK CHANGESITE-DIRECTED MUTAGENESIS KIT (Strategene) according to themanufacturer's protocols. Sequencing of the product verified that themutation was correctly introduced. X-1 bearing the mutation wasdesignated as mX-1, and the plasmid carrying mX-1 was designated as pmX-1 (FIG. 2).

Example 4 Construction of Chromosomal DNA Comprising OS-2 Type Mutation

Pα including exon 8 of the mouse PS-1 obtained in Example 1 was cleavedwith Nco I, and then treated with T4 DNA polymerase in the presence offour types of dNTPs to form blunt ends. The resulting fragment wasfurther cleaved with Asp718 I and then subjected to electrophoresis on1% agarose gel to collect an approximately 5-kbp DNA fragment includingexon 8. This fragment was ligated using T4 DNA ligase to the plasmidpBluescript II KS+which was cleaved beforehand with Sam I and Asp718 I,and then transformed into Escherichia coli to obtain a plasmid pSB-0.The plasmid pSB-0 was completely cleaved with Xba I, followed by partialdigestion with Pst I. Plasmid pmX-1 was cleaved with Xba I and Pst I andsubjected to electrophoresis on 1% agarose gel to collect mX-1. The mx-1was ligated to the Pst I fragment using T4 DNA ligase, and then used totransform Escherichia coli. The colonies of transformed E. coli werescreened to select a colony carrying a plasmid in which the X-1 portionin the plasmid pSB-0 was replaced with mX-1. The plasmid collected wasdesignated as pmSB-0 (FIG. 3).

Separately, Pα was cleaved with BamH I and Sal I and subjected toelectrophoresis on 1% agarose gel to collect an approximately 7-kbp DNAfragment including exon 8. This fragment was ligated using T4 ligase tothe plasmid pbluescript II KS+ which was cleaved beforehand with BamH Iand Sal I, and then used to transform E. coli to obtain plasmid pSB-1.The plasmid pmSB-0 was cleaved with Nco I and treated with T4 DNApolymerase in the presence of four types of dNTPs to form blunt ends.The resulting fragment was further cleaved with Xba I and subjected toelectrophoresis on 1% agarose gel. An approximately 2.2-kbp DNA fragmentXN including exon 8 was collected, and then the fragment and the plasmidpSB-1 were cleaved with Xba I and Pst I, and then the products weresubjected to electrophoresis on 1% agarose gel. The collectedapproximately 2.3-kbp DNA fragment PX not including exon 8 was ligatedusing T4 DNA ligase. The ligated fragment was further ligated using T4DNA ligase to the pBluescript II KS+ which was cleaved beforehand withXba I, blunt-ended with T4 DNA polymerase in the presence of four typesof dNTPs, re-ligated using T4 DNA ligase, and cleaved with Sma I and PstI. The resulting plasmid was subsequently transformed into E. coli. Thecolonies of transformed cells were screened to obtain plasmid pmSB-0′carrying only one DNA fragment in which DNA fragments XN and XP wereligated at the Xba I site (FIG. 4).

Example 5 Construction of Targeting Vector Backbone

To introduce an Eag I site into the Xba I site in plasmid pmSB-0′, anoligodeoxynucleotide having the following sequence was synthesized:

-   -   5′-CTAGACGGCCGT-3′ (SEQ ID NO: 21) (12 mer)

This oligodeoxynucleotide is capable of annealing via a nucleotidesequence having complementarity at the portion of CGGCCG, and formingthe following sequence after introduction at a site cleaved with Xba 1.

5′-TCTAGACGGCCGTCTAGA-3′ (SEQ ID NO: 22) 3′-AGATCTGCCGGCAGATCT-5′ (SEQID NO: 22)    Xba I Eag I Xba I

After the plasmid pmSB-0′ was cleaved with Xba I, the abovedeoxynucleotide was added to the product and ligated using T4 DNAligase, and then used to transform E. coli to obtain a plasmidpmSB-0′eag in which the Eag I site was inserted into the Xba I site ofthe plasmid pmSB-0′. After cleavage of pmSB-0'eag with Nco I and Sal I,resulting fragments were subjected to electrophoresis on 1% agarose gelto collect an approximately 5.3-kbp DNA fragment SN including exon 8.Separately, plasmid pSB-1 was cleaved with BamH I and Nco I and thensubjected to electrophoresis on 1% agarose gel to collect anapproximately 2-kbp DNA fragment NB not containing exon 8. The fragmentsSN and NB were ligated using T4 DNA ligase and treated with BamH I andSal I to obtain a DNA fragment in which both DNA fragments were ligatedat the Nco I site. This DNA fragment was further ligated to pBluescriptII KS+using T4 NDA ligase and then used to transform E. coli to obtain aplasmid pA (FIG. 5), wherein the pBluescript II KS+was cleavedbeforehand with Not I, blunt-ended with mung bean nuclease, re-ligatedusing T4 DNA ligase to break the Not I site and the Eag I siteoverlapping with the site, and cleaved with BamH I and Sal I.

Example 6 Construction of Targeting Vector

Plasmid pPNT (Victor L. J. et al., Cell Vol. 65, p. 1153, 1991) wascleaved with Xho I and BamH I and then treated with T4 DNA polymerase toform blunt ends and subjected to electrophoresis on 1% agarose gel. Thecollected approximately 1.7-kbp DNA fragment containing a neo expressionunit was ligated using T4 DNA ligase to the plasmid pBS246 (GIBCO BRL)which was cleaved beforehand with BamH I and treated with T4 DNApolymerase to form blunt ends, and then used to transform E. coli toobtain a plasmid pBS246neo. The plasmid was cleaved with Not I and thensubjected to electrophoresis on 1% agarose gel to collect anapproximately 2-kbp DNA fragment including the neo expression unitflanked by loxP sequences. The obtained DNA fragment was ligated usingT4 DNA ligase to the plasmid pA which was cleaved beforehand with Eag I,and then used to transform E. coli. Colonies of transformed cells werescreened to obtain plasmid pB in which the neo gene and the PS-1 genewere oriented in the same direction (FIG. 6).

After cleavage of the plasmid pB with BamH I and Sal I, the resultingfragments were subjected to electrophoresis on 1% agarose gel to collecta DNA fragment C containing the OS-2 type mutation and the neoexpression unit flanked by loxP sequences. Similarly, Pα was cleavedwith Sal I and BamH I, and the resulting DNA fragment of approximately6.5 kbp was subcloned into pbluescript II KS+ to construct a plasmidpSB-2, which was then cleaved with Hind III and BamH I and subjected toelectrophoresis on 1% agarose gel to collect a DNA fragment D ofapproximately 4 kbp. DNA fragments C and D were ligated using T4 DNAligase, and then the product was cleaved with Hind III and Sal I toobtain a DNA fragment in which C and D were ligated at the BamH I site.The obtained DNA fragment was further ligated using T4 DNA ligase to thepBluescript II KS+which was cleaved beforehand with Hind III and Sal I,and then used to transform E. coli to obtain a targeting vectorpOS-2neoloxP (FIG. 7).

Example 7 Introduction of Targeting Vector into ES Cells

Hereinafter in the examples, culture was carried out in an incubator at37° C. under 5% CO₂. The targeting vector was introduced byelectroporation into ES cells (R1) which were maintained in DMEM mediumsupplemented with 15% FBS and 10³ units/ml LIF (ESGRO) (the DMEM mediumis hereinafter abbreviated as ES medium). Culture medium was replacedwith fresh ES medium one day before electroporation, and the R1 cellswere collected and washed with electroporation buffer (20 mM HEPES, pH7.05, 137 mM NaCl, 5 mM KCl, 0.7 mM Na₂HPO₄, 6 mM dextrose). R1 cells(10⁷ cells) were mixed with 25 μg of the targeting vector pOS-2neoloxP,which was linearized using Not I, and 0.8 ml electroporation buffer inan electroporation cuvette. After 1 to 2 minutes, pulses were applied tothe cells using Bio-Rad GenePulser (Bio-Rad) under pulse conditions of240 V and 500 μF. The ES cells were collected by centrifugation andsuspended in 30 ml ES medium. The ES cell suspension (2 ml) was put ineach 10 ml culture dish in which feeder cells were put in 8 ml ESmedium. G418 (titer, 150 μg/ml) was added to the culture after 12 to 18hours, followed by one-week culture. As the feeder cell, a fibroblastestablished by the present inventors was used which was isolated from anembryo of 12 to 13 days obtained by mating a HS1 knockout male mouse (I.Taniuchi et al., EMBO J, vol. 14, p. 3664, 1995) with an ICR femalemouse of wild-type.

Example 8 Isolation of ES Cells with Homologous Recombination

Colonies of ES cells that were formed in Example 7 by one-weekcultivation after the addition of G418 were collected. Each colony wasdivided into two portions. One portion was subjected to furthercultivation. For selection of clones in which homologous recombinationoccurred, the other portion was washed with PBS, treated with ProteinaseK, and then chromosomal DNA was collected and subjected to PCR to selectclones. Nucleotide sequences of the synthetic primers used in PCRreaction were as follows.

-   -   Prsn1–2: 5′-CCCAACTCTATTTCTACCCTCGTTCATCTG-3′ (SEQ ID NO: 11)        (nucleotide sequence outside the targeting vector constructed)    -   PKG-1: 5′-TAGTGAGACGTGCTACTTCCATTTGTCACG-3′ (SEQ ID NO: 12)        (nucleotide sequence in the neo expression unit)

PCR reaction was carried out for 35 cycles under the followingconditions: 30 seconds at 93° C., 1 minute at 60° C., and 3 minutes at68° C. per cycle. The PCR product was analyzed by 1% agarose gelelectrophoresis to identify a positive clone which gave a band at anexpected position. The clone evaluated as positive was further subjectedto PCR using oligodeoxynucleotides PRL-101 and PRL-102. The resultingPCR product was cleaved with Sau3A I and then subjected toelectrophoresis on 2% agarose gel. Introduction of the mutation wasverified by split bands, and ES cells in which desired homologousrecombination occurred were selected. Nucleotide sequences of PRL-101and PRL-102 were as follows.

-   -   PRL-101: 5′-TGCTGGAGGAAAATGTGTTATTTAAGAGCA-3′ (SEQ ID NO: 13)    -   PRL-102: 5′-TACTGAAATCACAGCCAAGATGAGCCATGC-3′ (SEQ ID NO: 14)

Example 9 Production of Knockin Mouse

ES cells verified to have homologous recombination were further culturedfor 4 days and then treated with trypsin to separate one another. Aneight-celled embryo was taken from a BDF1 female mouse which was matedwith a BDF1 male mouse, and its zona pellucida was then removed. The EScells separated from one another were attached to the naked embryo (20ES cells per 8-celled embryo). Th treated embryo was transferred intothe uterus of a pseudopregnant female mouse and embryonic developmentwas continued to produce chimeric mice. The resulting chimeric malemouse was mated with a C57BL/6 female mouse. From among their progeny,mice with agouti color were chosen. A portion of the tail was excised,and chromosomal DNA was extracted from each sample. PCR was carried outusing PRL-101 and PRL-102, and the PCR product was cleaved with Sau3A Iand then subjected to electrophoresis on 2% agarose gel. The presence ofcleaved bands was examined to verify that the selected progeny possessedthe OS-2 type mutation. One male mouse was chosen from the verified miceand designated as #2.

Example 10

The knockin mouse #2 obtained in Example 9 has the heterozygous neoexpression unit flanked by loxPs deriving from the targeting vector.This mouse #2 (male, about 4 months old) was mated with a F4 female ofCAG-cre#13 transgenic mouse (2 months old in which transferred cre geneis heterozygous state, K. Sakai et al., Biochem. Biophys. Res. Commun.217:318, 1997). PCR was carried out using oligodeoxynucleotides PRL-100,PRL-102 and PGK-1 under the conditions described in Example 8. A mousefrom which the neo expression unit was removed was chosen as an OS-2mutated knockin mouse without the neo expression unit (FIG. 8). Thismouse was heterozygous with reference to OS-2 type mutation, and had oneloxP. Nucleotide sequences of PRL-100, PRL-102, and PGK-1 used for thePCR were as follows.

-   PRL-100: 5′-GGT CCA TCC CAG CTT CAC ACA GAC MG TCT-3′ (SEQ ID NO:    15)-   PRL-102: 5′-TAC TGA AAT CAC AGC CM GAT GAG CCA TGC-3′ (SEQ ID NO:    16)-   PKG-1: 5′-TAG TGA GAC GTG CTA CTT CCA TTT GTC ACG-3′ (SEQ ID NO: 17)

INDUSTRIAL APPLICABILITY

The gene-mutated animal of the present invention has a mutatedpresenilin-1 gene and high productivity of amyloid β due to the gene incomparison with a normal animal without the mutation, and hence theanimal exhibits symptoms of Alzheimer's disease through early cell-deathor deciduation of neurons in the cerebral hippocampus. Therefore,screening of substances useful for preventive and/or therapeutictreatment of Alzheimer's disease and evaluation of usefulness thereofcan be conducted by using the gene-mutated animal of the presentinvention.

1. A knockin gene-mutated mouse having a mutant presenilin-1 gene,wherein the mutant presenilin-1 gene results in overexpression ofAmyloid β 42 in the brain of said mouse.
 2. The gene-mutated mouseaccording to claim 1, wherein the animal has a mutant presenilin-1 genewhich comprises a DNA having a sequence encoding a presenilin-1 proteinin which an amino acid in the amino acid sequence of the presenilin-1protein is substituted with a different amino acid.
 3. The non humangene-mutated mouse according to claim 1, wherein the mouse has themutant presenilin-1 gene wherein a DNA sequence encoding around an aminoacid at position 213 in an amino acid sequence of the presenilin-1protein is mutated to the following sequence: 5′-TGTGGTCGGGATGATYGCC AVCCACTGGAAAGGCCC-3′ (SEQ ID NO: 18) wherein V represents a base other thanT, Y represents T or C, and the underlined bases encode the amino acidat position
 213. 4. The non human gene-mutated mouse according to claim1, wherein the mouse has the mutant presenilin-1 gene wherein a DNAsequence encoding around an amino acid at position 213 in an amino acidsequence of the presenilin-1 protein is mutated to the followingsequence: 5′-TGTGGTCGGGATGATMGCC ACC CACTGGAAAGGCCC-3′ (SEQ ID NO: 19)wherein Y represents T or C, and the underlined bases encode the aminoacid at position
 213. 5. The gene-mutated mouse according to claim 1,wherein the mouse has the mutant presenilin-1 gene wherein a DNAsequence encoding around an amino acid at position 213 in an amino acidsequence of the presenilin-1 protein is mutated to the followingsequence: 5′-TGTGGTCGGGATGATYGCC NNN CACTGGAAAGGCCC-3′ (SEQ ID NO: 20)wherein each N independently represents A, G, T, or C and NNN representsa codon as triplet bases which encodes an amino acid other thanisoleucine, Y represents T or C, and the underlined bases encode theamino acid at position
 213. 6. The non human gene-mutated mouseaccording to claim 1, wherein the mutant presenilin-1 gene istransferred by homologous recombination.
 7. A knockin gene-mutated mousehaving a mutant presenilin-1 gene which comprises a DNA having asequence encoding a mutant mouse presenilin-1 protein in whichisoleucine at position 213 of a mouse presenilin-1 protein as set forthin SEQ ID NO: 3 is substituted with an amino acid other than isoleucine,wherein the mutant presenilin-1 gene results in overexpression ofAmyloid β 42 in the brain of said mouse.
 8. A knockin gene-mutated mousehaving a mutant presenilin-1 gene which comprises a DNA having asequence encoding a mutant mouse presenilin-1 protein in whichisoleucine at position 213 of a mouse presenilin-1 protein as set forthin SEQ ID NO: 3 is substituted with threonine, wherein the mutantpresenilin-1 gene results in overexpression of Amyloid β 42 in the brainof said mouse.
 9. A gene-mutated mouse having a mutant presenilin-1 genewhich encodes for the OS-2 type mutation of presenilin-1, wherein themutant presenilin-1 gene results in overexpression of Amyloid β42 in thebrain of said mouse.
 10. A method for evaluating the therapeutic effector preventive treatment of a substance on Alzheimer's disease, whichcomprises: administering a test substance to a gene-mutated mouseaccording to claim 1, then determining a total amount of amyloid β inthe brain (M) and the amount of amyloid β 40 and amyloid β 42 in thebrain, then calculating a ratio of amyloid β 42/amyloid β 40 (P);administering a reference substance to a gene-mutated mouse according toclaim 1, then determining a total amount of amyloid β in the brain (N)and the amount of amyloid β40 and amyloid β 42 in the brain, thencalculating a ratio of amyloid β 42/amyloid β40 (Q); and comparing thevalue of M to N, or the value of P to Q.
 11. The method for evaluationaccording to claim 10, wherein the comparison is conducted for one ormore items selected from the group consisting of survival period oftime, exploratory behavior, and migratory behavior.
 12. A primary cellculture or a subcultured cell obtainable by isolating a cell from thegene-mutated mouse according to claim 1 and culturing said cell bytissue culture.
 13. A method for evaluating a medicament for therapeuticand/or preventive treatment of Alzheimer's disease which comprises thestep of culturing the primary cell culture or the subcultured cellaccording to claim 12 in vitro in the presence of a test compound. 14.An embryo introduced with a plasmid comprising a DNA represented by thenucleotide sequence: 5′-TGTGGTCGGGATGATYGCCACCCACTGGAAAGGCCC-3′ (SEQ IDNO: 19) wherein Y represents T or C.
 15. An embryo obtained byhomologous recombination using the plasmid according to claim
 1. 16. Amethod for producing a gene-mutated mouse, wherein the method comprisesthe step of transferring a mutant presenilin-1 gene by homologousrecombination into an embryo of a mouse, wherein the mutant presenilin-1gene is capable of expressing a mutant presenilin-1 protein and inducingproduction of amyloid β protein in an amount sufficient to form aprogressive neural disease in the hippocampus or a peripheral portion ofthe cerebral cortex of the brain.
 17. The method according to claim 16,wherein the gene mutated presenilin-1 mouse is capable of expressing amutant presenilin-1 protein in which isoleucine at position 213 of amouse presenilin-1 protein as set forth in SEQ ID NO: 3 is substitutedwith an amino acid other than isoleucine.